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Multicenter validation of targeted NGS in CLL
Supplementary Appendix). Overall, none of the four variants with a VAF <5% reported by a single institute using the Multiplicom assay were found by any other center utiliz- ing the Illumina or HaloPlex methodologies; further sup- porting the notion that they are false positive results (Online Supplementary Table S10).
Pairwise comparison of the data generated from the centers utilizing the Illumina assay revealed that 128 vari- ants were detected and could subsequently be assigned to the groups defined above as follows i.e., group 1 (n=100), group 2 (n=6), group 3 (n=19), group 4 (n=0) and group 5 (n=3) (Figure 2; Online Supplementary Table S9, S11). The concordance rate was 97.7% (125 of 128) with three vari- ants detected by only one center; all three variants had a VAF <5% (Online Supplementary Table S9-10). The finding of 2 of 3 of these variants in the centers utilizing the other technologies portends to them being true subclonal muta- tions present in a minor proportion of leukemic cells (Online Supplementary Table S9).
A total of 127 variants were found using the HaloPlex assay; however, as three samples failed the sequencing run in center 5 (T28, T41 and T48) which collectively har- bored eight variants, these samples (and their variants) were excluded from the pairwise analysis. The remaining 119 variants were distributed amongst the various groups as follows: group 1 (n=89), group 2 (n=5), group 3 (n=13), group 4 (n=7) and group 5 (n=5) (Figure 2; Online Supplementary Tables S9, S12). The concordance rate was 90% (107 of 119) with 12 variants detected by only one center; 7 of these 12 variants had a VAF >5% with the remaining 5 variants having a VAF <5%. Investigating the variants with a VAF > 5% (n=7) revealed that (i) 3 of these variants were found in all other centers indicating that they were false-negatives for center 6; (ii) two variants in T5 could be false-positives for center 5 (discussed in more detail in the Online Supplementary Appendix); and, (iii) for the remaining two variants (VAF 10% and 6.7%), one was not found by any other center while the latter was found at a very low frequency of 0.56% (Online Supplementary Table S9-10). Notably, the five variants found with a VAF <5% were all detected by the other methodologies.
Overall, the VAF reported were in agreement between partner centers and this similarity was evidenced by the narrow range of VAF for a particular variant, thereby indi- cating the accuracy of each assay in reporting VAF (Figure 2).
NFKBIE and EGR2 were only included in the Illumina and HaloPlex gene panel designs. Within the HaloPlex dataset a total of six mutations were reported in the NFKBIE gene, five of which were found in both institutes at a VAF >5%, with the remaining variant found either above or below 5% in the partner center (Online Supplementary Table S13). Thus, the concordance rate for NFKBIE mutations detected using the HaloPlex assay was 100%. A total of seven NFKBIE mutations were found by the Illumina assay. However, while four were concordant, being found in both centers and at a VAF >5%, the remaining three variants were only found by a single cen- ter (two with a VAF >5% and the remainder with a VAF <5%) (Online Supplementary Table S13). Two of the three variants were found by the centers performing the analy- sis with the HaloPlex technology, indicating that these mutations are unlikely to be false-positives in the single Illumina center and false-negatives in the second center is the more probable explanation.
Finally, the HaloPlex assay detected a total of five vari- ants within the EGR2 gene; three variants were found with a VAF >5% by both centers while two variants were found by both centers with a low VAF (<5%) (Online Supplementary Table S13). Taken collectively, the concor- dance for EGR2 mutation detection reached 100% for the HaloPlex assay. In contrast, EGR2 proved to be the most difficult gene in this study to sequence when using the Illumina assay. Four variants were detected using the Illumina panel, however, only one was found by both cen- ters, which, surprisingly, concerned a low frequency vari- ant (VAF 2.3% and 2.2%) (Online Supplementary Table S13). Notably, two of the variants found by Illumina were not present in the HaloPlex datasets. This dropout of ampli- cons for EGR2 is supported by the coverage data (Online Supplementary Table S6). While reaching a definitive con- clusion as to why this dropout occurred is not possible, drawing from our previous experience, reagents based on the older TSCA chemistry had a relatively short period within which they performed optimally i.e., while well covered regions maintained a high coverage when using reagents nearing or older than 3 months, the coverage for problematic regions such as the NOTCH1 PEST domain, NFKBIE, EGR2 and certain regions within the TP53 gene decreased dramatically.
Inter-laboratory variation in detecting mutations
We next assessed the reproducibility of targeted NGS by looking at the inter-laboratory variation in detecting mutations. As these assays were not specifically designed to detect variants with a very low VAF i.e., <5%, filtering was set such that a VAF of 5% had to be reached in at least one center for the variant to be included in the concor- dance counts. We found that 107 of 115 (93% concor- dance) mutations were consistently detected by all six par- ticipating centers (Online Supplementary Table S9). These 107 variants could be segregated into two groups: (i) 87 variants (81%) were found at a frequency >5% in all six centers; and, (ii) 20 variants (18.7%) were bordering the 5% cut-off, and, with the exception of sample T5, their VAF spanned a narrow range (1-12%) (Online Supplementary Table S9). This is illustrated in Figure 3 for variants detected within TP53, SF3B1, and NOTCH1. The remaining 8 of 115 (7%) variants were not detected by a single center i.e., the variants were successfully found in the sequencing data from 5 of 6 of the participating cen- ters. Two of these variants went undetected in centers uti- lizing the HaloPlex technology and concerned frameshift insertions within the ATM gene (p.L1327fs [center 5] and p.V2201fs [center 6]) (Online Supplementary Table S9). Analysis of the raw sequencing data revealed that ampli- con dropout leading to a gap in coverage resulted in these undetected ATM frameshift mutations. The final six muta- tions were present at a low frequency and were not found within the following centers sequencing datasets, center 4 (n=1), center 5 (n=2) and center 6 (n=3). These minor sub- clonal mutations were detected by the partner institute utilizing the same methodology, thus demonstrating the ability of the assay to detect and amplify in this genomic region.
Finally, a variant was deemed as a false-positive if it was only found in a single test center. Overall, four false-posi- tives with a VAF ≥5% were detected in two centers and analysis of these variants led to the conclusion that it is highly probable that 3 of these 4 false-positive calls arose
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